All cells are subject to and respond to mechanical forces like compression. However the molecular mechanisms linking the mechanics to biological responses are not fully understood. The cells of our model system, the chondrocytes of cartilage, undergo compression in vivo, and these cells can transduce compression into biological signals. There is evidence that glucose utilization in chondrocytes is regulated by compression and that physiologic compression stimulates glycolysis, the main pathway chondrocytes use to make ATP. This phenomenon has been linked to the ability of chondrocytes to maintain cartilage. Thus, the study of glucose metabolism is relevant to NIH because millions suffer from chondrocyte-driven cartilage deterioration in osteoarthritis. Current osteoarthritis treatments involve joint motion, which is counterintuitive. We show for the first time that physiologically relevant culture conditions enable in vitro compression of chondrocytes. This project tests the hypothesis that physiological compression of both normal and osteoarthritic chondrocytes results in a specific pattern of metabolites within glucose metabolism that support protein production to maintain the cellular microenvironment. The premise is that by quantifying glucose metabolism in chondrocytes this project will develop strategies that use mechanical loading to produce the building blocks for cartilage repair. Aim 1 - In vitro experiments will examine the source of carbon (glucose or glutamine) and the mechanism of regulation. Dependent variables include sex, donor age and the level (low or high) of applied compression. Targeted metabolomics data will be generated from normal and osteoarthritic chondrocytes subjected to compression under different experimental conditions. Aim 2 - Experiments using mice subjected to voluntary running will assess in vivo mechanotransduction. Dependent variables include sex and the duration of running. Readouts will include both targeted metabolites and immunohistological markers examining regulation of glucose metabolism. Assays will employ highly specific enzyme inhibitors that will allow a step-by-step analysis of critical metabolic pathways. This project has substantial innovation including a novel systems biology model and analytical approach that calculate the relative rates of reaction for each step in glucose metabolism. These modeling results will be used both to refine existing hypotheses and to generate new ones. The goal of this project is to identify changes in patterns of small metabolites that result from compression for normal and osteoarthritic chondrocytes. The expected outcome is to identify candidate target reactions that leverage glucose metabolism to increase mechanically driven production of amino acid precursors to repair cartilage. Understanding these mechanisms may prove useful in developing translational strategies to heal cartilage by activating existing mechanosensitive pathways. Insight into how chondrocytes respond to compression will advance osteoarthritis translation by providing new therapeutic targets for cartilage repair and enabling substantial clinical progress.